1. Field of Invention
The invention relates to methods for generating bioluminescence-labeled Gram-negative bacteria in order to overcome the drawback of the difficulty to tracking the bacteria inside their hosts, because it offers a powerful tool to tracking Gram-negative bacteria in vivo using a stably and highly bioluminescence expressing plasmid vehicle.
2. Description of Related Art
For observing bacterial distribution and behavior inside the animal body, it conventionally needs the sacrifices of the experimental animals and the analyses of animal organ specimens. Therefore, an ideal method using light-emitting (bioluminescent) gene expression system of bacterial luxABCDE has been developed to observe the dynamic changes of bacterial distribution and behavior without animal sacrifice while bacteria existing inside their host bodies.
Although many methods have been previously provided to study the bacterial behavior and distribution inside their host bodies using light-emitting gene expression in bacteria, there are still certain drawbacks to limit their applications. They are as the follows: (i) the plasmids used to express the bioluminescence could not stably exist in bacteria without any selection stress, as a result of plasmid loss after couple generation; (ii) the delivery method, such as electroporation or competence, is common to transfer plasmid into bacterium, and however, it is restricted by bacterial capsule, which is composited of polysaccharides and can be a crucial barrier to limit the bacterial transformation to very low rate; (iii) the transposons utilized to insert the bioluminescence gene marker into bacterial chromosome usually transpose randomly into uncertain transposition site with unacceptably low transposition rate, and therefore the resulting individuals are different, and difficult to select and to confirm whether their insertion sites are crucial for further characteristic analysis; and (iv) the double crossing-over replacement applied to insert a marker at a specific site in chromosome needs many tedious cloning, and worse, its replacement efficiency is very poor.
Additionally, the bioluminescence genes obtained via transposition or gene replacement are just a single copy in bacterial chromosome, which might not be expressed as highly as in a high copy number of plasmid, such as the plasmid containing ColE1 replication origin. Thus, the need for improvement still exists.